Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics.
Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics.
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DOI：
10.1126/science.abf8761
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发表时间：
2021-07-23
期刊：
影响因子：
--
通讯作者：
Fordyce PM
中科院分区：
文献类型：
--
作者：
Markin CJ;Mokhtari DA;Sunden F;Appel MJ;Akiva E;Longwell SA;Sabatti C;Herschlag D;Fordyce PM
酶具有非凡的催化精确度和专一性。这些性质最终不仅来自活性中心残基和底物之间的相互作用，而且来自整个折叠酶的功能相互作用。因此，理解催化精确度和特异性的起源将需要在整个蛋白质中进行突变的能力。传统上，酶活性位点是通过定点突变(SDM)来表征的，这揭示了这些残基的催化功能；然而，SDM是低通量、昂贵和劳动密集型的。相比之下，最近发展起来的高通量突变扫描技术检测了大量的序列，但只提供了功能的粗略估计，例如在特定条件下特定时间产生的产生量或整体生物体适应性。需要新的技术来克服目前方法的局限性，并允许以成本和时间高效的方式对许多酶变体进行深入表征。为了应对这一挑战，我们开发了一个高通量微流控平台，允许在几个小时内同时表达和纯化1500多个合理选择的酶突变，并允许在几天内对它们的功能进行定量鉴定。HT-MEK(高通量微流控酶动力学)可以与任何可在体外标记和表达的酶系统一起使用，并具有直接或耦合的荧光分析。作为HT-MEK的第一次应用，我们对1036个单位点突变体进行了功能鉴定，这些突变体在PAFA(黄杆菌磷酸抑制碱性磷酸酶)中的每个位置都包含甘氨酸或缬氨酸取代，PAFA是碱性磷酸酶超家族中一种研究得很好的酶。对于每个突变体，我们测量了多底物的Michaelis-Menten动力学[表观单分子速率常数(Kcat)、Michaelis常数(Km)和kcat/Km]、抑制常数和对折叠的影响，从670,000多个反应中获得了5000多个动力学和热力学常数。我们发现，大多数突变(1036个中的702个)在催化的某些方面产生了统计上显著的影响。通过系统和独立地改变表达和检测条件，我们确定了232个突变通过促进形成长期的、催化不活跃的错误折叠状态来降低催化作用，而在我们的检测条件下，没有一个突变是通过平衡展开来降低催化作用的。将这些功能测量与先前的机制知识相结合，使我们能够系统地评估每个突变的影响。不同的残基组影响功能的不同方面，影响特定功能的残基形成了从活性部位到活性部位到酶表面的大的空间连续区域，从活性部位到酶表面。HT-MEK使我们能够揭示整个PAFA的功能效应，并确定不同残基对催化特性的影响。这些影响中的一些很容易通过检查与活性部位的结构互连而合理化，而另一些则不明显，包括大的远端和表面效应以及发现长期错误折叠状态。这些结果强调了在多个反应条件下测量突变对多个动力学和热力学参数的影响的必要性，因此需要这项新技术。由于HT-MEK适用于任何具有直接或耦合荧光读数的酶，并以较低的成本快速地提供了对突变空间的深入和定量分析，因此它可能是表征新酶的首选方法。在未来的应用中，HT-MEK可用于剖析潜在的进化轨迹，确定人类疾病相关等位基因变异的功能后果，识别具有合理控制催化的新生变构潜力的表面，并指导天然和设计的酶适应新的功能和角色。在微流控设备中同时表达、纯化和生化鉴定酶变异体，使得在几天内测量1500多个变异体的米氏参数和抑制常数成为可能。多种分析的突变效应揭示了一个广泛的功能结构，在这个结构中，物理上相邻的残基区域延伸到酶表面，控制或改变催化的特定方面。需要对酶进行系统和广泛的研究，以了解它们的非凡效率，并应对当前医学和工程领域的挑战。我们提出了高通量微流控酶动力学(HT-MEK)，这是一个用于每个实验高通量表达、纯化和鉴定1500多种酶变体的微流控平台。对于1036个突变的碱性磷酸酶PAFA(黄杆菌的磷酸盐抑制型碱性磷酸酶)，我们进行了超过670,000个反应，并测定了超过5000个对多种底物和抑制剂的动力学和物理常数。我们揭示了广泛的动力学分配到错误折叠状态和孤立的催化效应，揭示了与功能的特定方面相关的空间上连续的残基区域。区域包括活性部位的近端残基，但延伸到酶表面，提供了现有方法不可能得出的潜在结构图。HT-MEK的应用范围从了解分子机制到医学、工程和设计。
Enzymes possess extraordinary catalytic proficiency and specificity. These properties ultimately derive from interactions not just between the active-site residues and the substrate but from functional interactions throughout a folded enzyme. Therefore, understanding the origins of catalytic proficiency and specificity will require the ability to make mutations throughout the protein. Traditionally, enzyme active sites have been characterized by means of site-directed mutagenesis (SDM), revealing much about the catalytic functions of these residues; nevertheless, SDM is low-throughput, costly, and labor intensive. By contrast, recently developed high-throughput mutational scanning techniques assay large numbers of sequences but provide only coarse estimates of function, such as the amount of product generated at a particular time under a particular set of conditions or overall organismal fitness. New technologies are needed to overcome the limitations of current approaches and allow deep characterization of many enzyme variants in a cost- and time-efficient manner. To meet this challenge, we developed a high-throughput microfluidic platform that allows the simultaneous expression and purification of more than 1500 rationally chosen enzyme mutants in hours and allows their quantitative functional characterization in days. HT-MEK (High-Throughput Microfluidic Enzyme Kinetics) can be used with any enzyme system that can be tagged and expressed in vitro and has a direct or coupled fluorogenic assay. As a first application of HT-MEK, we functionally characterized 1036 single-site mutants that contain either a glycine or valine substitution at each position within PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), a well-studied enzyme from the alkaline phosphatase superfamily. For each mutant, we measured Michaelis-Menten kinetics [apparent unimolecular rate constant (kcat), Michaelis constant (Km), and kcat/Km] for multiple substrates, inhibition constants, and effects on folding, obtaining more than 5000 kinetic and thermodynamic constants from more than 670,000 total reactions. We found that most mutations (702 of 1036) yielded statistically significant effects on some aspect of catalysis. By systematically and independently varying expression and assay conditions, we determined that 232 of these mutations reduced catalysis by promoting the formation of a long-lived, catalytically inactive misfolded state, whereas none did so through equilibrium unfolding under our assay conditions. Combining these functional measurements with prior mechanistic knowledge allowed us to systematically assess the effect of each mutation. Different groups of residues affected different aspects of function, with residues that affect a particular function forming large, spatially contiguous regions that spanned from the active site up to 20 Å from the active site and to the enzyme surface. HT-MEK has allowed us to uncover functional effects throughout PafA and to identify the catalytic features affected by different groups of residues. Some of these effects are readily rationalized through inspection of structural interconnections to the active site, whereas others were nonobvious, including large distal and surface effects and the discovery of a long-lived misfolded state. These results underscore the need to measure the effects of mutations on multiple kinetic and thermodynamic parameters across multiple reaction conditions and thus the need for this new technology. Because HT-MEK is applicable to any enzyme with a direct or coupled fluorescent readout and provides an in-depth and quantitative analysis of mutant space rapidly and at modest cost, it may be the method of choice to characterize new enzymes. In future applications, HT-MEK can be used to dissect potential evolutionary trajectories, determine the functional consequences of human disease-associated allelic variants, identify surfaces with nascent allosteric potential for rational control of catalysis, and direct the adaptation of natural and designed enzymes for new functions and roles. Simultaneous expression, purification, and biochemical characterization of enzyme variants in a microfluidic device makes it possible to measure Michaelis-Menten parameters and inhibition constants for more than 1500 variants in days. Mutational effects across multiple assays reveal an extensive functional architecture in which physically contiguous residue regions extending to the enzyme surface control or alter particular aspects of catalysis. Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.
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影响因子：
64.5
作者：
CARTER, PJ;WINTER, G;FERSHT, AR
通讯作者：
FERSHT, AR
影响因子：
14.8
作者：
Dagliyan, Onur;Dokholyan, Nikolay, V;Hahn, Klaus M.
通讯作者：
Hahn, Klaus M.
影响因子：
2.9
作者：
ALBERY, WJ;KNOWLES, JR
通讯作者：
KNOWLES, JR
DOI：
10.1073/pnas.1417811112
发表时间：
2015-03-03
影响因子：
11.1
作者：
Bowman, Gregory R.;Bolin, Eric R.;Marqusee, Susan
通讯作者：
Marqusee, Susan
影响因子：
7.2
作者：
Gidalevitz, Tali;Prahlad, Veena;Morimoto, Richard I.
通讯作者：
Morimoto, Richard I.